Berberine regulates UCP1 expression by reshaping the conformation of quadruplex formed by the element of the UCP1 gene promoter

Obesity has become a serious issue due to its worldwide prevalence and is the leading risk factor for other metabolic diseases. Increasing evidence indicates that the activation and recruitment of brown adipose tissue (BAT) and the induction of the expression of uncoupling protein 1 (UCP1) are attractive strategies to increase metabolic efficiency and counteract weight gain [1]. UCP1, a thermogenic protein, is located in the inner mitochondrial membrane, is mainly expressed in brown and beige/brite adipose tissues and plays critical roles in metabolic and energy balance by releasing chemical energy as heat [2]. Furthermore, the activity of the UCP1 promoter has been shown to be activated during the browning of white adipose tissue (WAT) and is generally inactivated in tissues except brown and beige/brite adipose tissues [3]. Therefore, targeting the activity of the UCP1 gene promoter might be a promising strategy to regulate the expression of UCP1. G-quadruplexes are nucleic acid secondary structures formed in G-rich sequences in DNA or RNA, which include single nucleic acid strands (unimolecular G-quadruplex, Uni-G4), between strands (bimolecular G-quadruplex, Bi-G4), and tetramolecular quadruplexes (tetra-G4). G-quadruplexes have

*Correspondence address. Tel: +86-23-62563182; E-mail: fyin@cqut.edu.cn Obesity has become a serious issue due to its worldwide prevalence and is the leading risk factor for other metabolic diseases. Increasing evidence indicates that the activation and recruitment of brown adipose tissue (BAT) and the induction of the expression of uncoupling protein 1 (UCP1) are attractive strategies to increase metabolic efficiency and counteract weight gain [1]. UCP1, a thermogenic protein, is located in the inner mitochondrial membrane, is mainly expressed in brown and beige/brite adipose tissues and plays critical roles in metabolic and energy balance by releasing chemical energy as heat [2]. Furthermore, the activity of the UCP1 promoter has been shown to be activated during the browning of white adipose tissue (WAT) and is generally inactivated in tissues except brown and beige/brite adipose tissues [3]. Therefore, targeting the activity of the UCP1 gene promoter might be a promising strategy to regulate the expression of UCP1.
G-quadruplexes are nucleic acid secondary structures formed in Grich sequences in DNA or RNA, which include single nucleic acid strands (unimolecular G-quadruplex, Uni-G4), between strands (bimolecular G-quadruplex, Bi-G4), and tetramolecular quadruplexes (tetra-G4). G-quadruplexes have become promising therapeutic targets due to their roles in the expressions of many critical genes. There is a G-quadruplex-forming sequence in the promoter of the UCP1 gene, and mutation in the G-quadruplex sequence dramatically enhances the activity of the UCP1 gene promoter, suggesting that the G-quadruplex is a potential target to regulate UCP1 expression [4].
Berberine has been shown to induce the development of beige/ brite adipocytes by increasing the expression of UCP1 and proved to have metabolic benefits by increasing energy expenditure and limiting weight gain in obese and/or db/db mice [5,6]. Although the roles of transcription cascades and epigenetics in the regulation of the activity of the UCP1 gene promoter have been widely reported, it is not clear whether berberine affects the activity of the UCP1 gene promoter and its associated mechanisms.
In this study, we characterized the effects of berberine on the Gquadruplex formed by the oligonucleotide fragment of the UCP1 gene promoter (Olig) using a variety of spectroscopic techniques (UV-visible, fluorescence, and circular dichroism) and native gel electrophoresis approaches. We also determined the influence of berberine on the expression of the UPC1 gene and the biosynthesis of fatty acids in differentiated 3T3-L1 adipocytes.
The oligonucleotide fragment of the UCP1 gene promoter (CGAGGGTGGGTAGGAGGGGACGCGGGGACT, Olig) and its G→A mutated sequence (CGAGGGTAAATAGGAAAAAACG-CAAAAACT, muOlig) were synthesized by Sangon Biotech (Shanghai, China), dissolved in double-distilled water, and prepared in 10 mM Tris-HCl buffer (pH 7.4). After the DNA samples were heated to 95°C for 5 min, the indicated dosages of berberine and/or KCl were mixed with the DNA samples before the DNA was slowly cooled to room temperature. Absorption spectroscopy was recorded using a Shimazu 2450 UV-vis spectrophotometer at 25°C from 280 to 550 nm. As shown in Figure 1A, there were two distinct peaks of berberine solution at 340 and 420 nm; the addition of quadruplex caused a significant hypochromicity (approximately 45%) and a moderate bathochromic shift of 8 nm for the high-energy peak from 340 to 348 nm and hyperchromicity with a redshift of 50 nm for the low-energy peak from 420 to 470 nm. These data suggest that berberine binding with oligonucleotide fragments results in hypochromicity and bathochromism through a strong intercalation between berberine and the G-quadruplex. At the same time, fluorescence spectroscopy was performed on a fluorescence spectrometer (Thermo Fisher Scientific, Waltham, USA) from 450 to 700 nm at an excitation wavelength of 280 nm at 25°C, and the excitation and emission slit widths were set as 5 and 10 nm, respectively. As shown in Figure 1B, berberine alone in Tris buffer was nonfluorescent, but in the presence of G-quadruplexes, the fluorescence intensity of berberine was increased dramatically, and the λ max in the fluorescence emission spectra shifted to the blue end by 8 nm. Moreover, the effect of berberine on the conformation of the G-quadruplex formed by Olig was further evidenced by CD spectroscopy, which was recorded on a spectropolarimeter (CHIR-ASCAN; Applied Photphysics Ltd, Leatherhead, UK) from 200 to 350 nm at 25°C in a 0.1 cm path length quartz cell at a scanning rate of 20 nm/min. As shown in Figure 1C, CD spectroscopy exhibited a characteristic peak of the G-quadruplex at 260 nm in the presence of potassium, a known G-quadruplex stabilizer [7], while berberine partly increased the intensity of the peak at 260 nm.
G-quadruplexes are compact structures, so they migrate faster than non-G-rich oligonucleotides with the same length [8]. To further distinguish the effects of berberine on the conformation of the Gquadruplex formed by Olig, we used native gel electrophoresis to observe the G-quadruplex structure with or without berberine. The results indicated that in the presence of 100 mM KCl, almost all the Gquadruplexes formed by Olig were Uni-G4, but berberine caused a transformation of G-quadruplexes from Uni-G4 to Tetra-G4 ( Figure 1D).
Mouse-derived 3T3-L1 preadipocytes, purchased from the China Center for Type Culture Collection (Wuhan, China), were induced to differentiate into adipocyte-like cells before being used by incubating with 0.5 mM isobutyl methylxanthine (IBMX, ab120840; Sigma, St Louis, USA) and 1 μM dexamethasone (Sigma) for 3 days. Then, the medium was replaced with complete media plus 10 μg/mL insulin (MCE, Monmouth Junction, USA), and the cells were cultured for another 3 days. Before determining the effect of berberine on the expression of UCP1, we initially evaluated the cytotoxicity of berberine in differentiated 3T3-L1 adipocytes by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay after the cells were cultured with the indicated doses of berberine for 24 h. The results demonstrated that, once the concentration of berberine was over 20 μM, berberine showed noticeable cytotoxicity (data not shown). Then, we determined the effect of berberine on the activity of the UCP1 gene promoter with a reporter gene assay. Generally, after the promoter of the human UCP1 gene was cloned and transfected into HEK293 cells using Lipofectamine 3000 (Invitrogen, Carlsbad, USA) and incubated for 48 h, the luciferase activity was determined with the

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Berberine, a novel regulator of UCP-1 Dual-Glo® Luciferase Assay System (Promega, Madison, USA) according to the supplier's suggestion. The data revealed that berberine treatment evidently increased the activity of the UCP1 gene promoter (Figure 2A). Furthermore, incubation with berberine increased the expression of UCP1 in a time-and dose-dependent manner ( Figure 2B-D), and treatment with 10 μM berberine for 6 h Berberine, a novel regulator of UCP-1 1759 increased the mRNA level of UCP1 by approximately 4.3-fold ( Figure 2B). Incubation with 10 μM berberine for 72 h increased the protein level of UCP1 by approximately 2-2.5 folds ( Figure 2C,D). Accumulating evidence indicates that the browning/beigeing of WAT can be leveraged to treat obesity due to its distinctive capacity to drive thermogenesis, and UCP1 is the best thermogenic effector in adipocytes, which uncouples respiration from ATP synthesis, dissipates the proton gradient as heat to maintain body temperature in a cold environment or wastes energy [9]. Meanwhile, BAT is rich in mitochondria and plays a potential role in energy expenditure and glucose homeostasis, and UCP1 has been used as a hallmark protein in the browning/beigeing of WAT [10].
To explore the actions of berberine that regulate the expression of UCP1 in adipocytes, differentiated 3T3-L1 adipocytes were cultured with 0, 2.5, 5 and 10 μM berberine for 48 h. After that, the cells were fixed with fresh 4% paraformaldehyde for 10 min and then stained with oil red O for 20 min. The results showed that treatment with berberine for 48 h decreased the accumulation of lipid droplets in a dose-dependent manner in differentiated 3T3-L1 adipocytes ( Figure 2E,F). Interestingly, incubation with berberine also dosedependently increased the content of mitochondria in differentiated 3T3-L1 adipocytes ( Figure 2G,H).
To clarify the mechanisms by which berberine attenuates the accumulation of lipid droplets in differentiated 3T3-L1 adipocytes, the expression of fatty acid synthesis (FAS) and acetyl-CoA carboxylase 1 (ACC1), the two key genes for the biosynthesis of fatty acids, were determined by qRT-PCR. Generally, the cells were incubated with 10 μM berberine for the indicated time, and total RNA was extracted using TRIzol reagent (Invitrogen). First-strand cDNA was generated using the random hexamer primer provided with an iScript cDNA synthesis kit (Bio-Rad, Hercules, USA). The qPCR experiments were conducted on a Step-One plus real-time PCR system and carried out using a final volume of 10 μL, containing 1 ng of reverse-transcribed total cDNA, 2 nM of forward and reverse primers, and SYBR green PCR master mixture, which was performed in 96-well plates using the CFX96 real-time PCR system (Bio-Rad). The PCR conditions consisted of 40 cycles with 15 s denaturation at 95°C, 30 s annealing at 55°C, and 60 s extensions at 72°C. The fold change in mRNA was calculated by the 2 -∆∆Ct method using β-actin as the reference gene to normalize the data for all samples. The specific primers were mACC1 (forward: 5′-GAATCTCCTGGTGACAATGCTTATT-3′, reverse: 5′-GGTCTTGCTGAGTTGGGTTAGCT-3′), mFAS (forward: 5′-TGGGTTCTAGCCAGCAGAGT, reverse: TACCACCAGAGACCGT TATGC-3′), and β-actin (forward: 5′-GGCTGTATTCCCCTCCATCG-3′, reverse: 5′-CCAGTTGGTAACAATGCCATGT-3′). Our data indicated that treatment with berberine significantly decreased the mRNA levels of FAS and ACC1 genes in differentiated 3T3-L1 adipocytes ( Figure 2I,J).
In summary, the spectroscopic characteristics demonstrated that in the presence of Olig, the solution of berberine showed a significant hypochromicity and a moderate bathochromic shift for the high-energy peak at 340 nm and hyperchromicity and a redshift of the low-energy peak at 420 nm. The fluorescence intensity of the berberine solution was also dramatically increased by the addition of quadruplexes formed by Olig. All of these spectroscopic data suggested that berberine might not be a good quadruplex DNA intercalator but rather a groove binder. Meanwhile, native gel electrophoresis indicated that berberine caused a significant transformation of G-quadruplexes from Uni-G4 to Tetra-G4. Furthermore, together with increasing the content of mitochondria, berberine treatment evidently increased the activity of the UCP1 gene promoter, induced the expression of UCP1 in a dose-and timedependent manner, and attenuated the accumulation of lipid droplets by reducing the expression of FAS and ACC1 in differentiated 3T3-L1 adipocytes. This study suggested that the metabolic benefits of berberine, by increasing energy expenditure and limiting weight gain in obese and/or db/db mice, could be involved in its role in the transformation of quadruplexes formed by the UCP1 gene promoter and its expression.